Our laboratory is investigating the molecular processes involved in developing a terminally differentiated organism from a homogeneous population of totipotent cells. We are using molecular, genetic, and biochemical techniques and the model eukaryotic system Dictyostelium discoideum to define cell autonomous and non- autonomous signal transduction pathways that specify cell fate and pattern formation. Our research serves to identify receptor-mediated cascades and nuclear events that may be central to metazoan differentiation. We have shown that the different cAMP receptor (CAR) subtypes in Dictyostelium act both cooperatively and antagonistically to promote or to inhibit cell- specific differentiation and axis formation. The high affinity receptors CAR1 and CAR3 are coupled to an activation pathway for GSK3 which is required for the differentiation of posterior (prespore) cells in the organism; the low affinity receptors CAR2 and CAR4 inhibit GSK3 activity which suppresses posterior differentiation, but promotes anterior cell (prestalk) differentiation. Thus, car4-nulls exhibit enhanced activation of GSK3 during development, as contrasted with the low level of GSK3 activity observed in car3-nulls. Further, car4-null strains and gsk3-null strains have essentially opposite developmental phenotypes. car4-nulls are inhibited in anterior (prestalk) differentiation, while gsk3-nulls have posterior defects. Many aspects of the car4-null phenotype are rescued by co-expression of a dominant-negative GSK3, supporting the upstream, inhibitory function for CAR4 control of GSK3 and subsequent anterior/posterior cell fate decisions. It is generally presumed that all 7-TM receptor signaling in the metazoa is mediated via heterotrimeric G proteins. Activation of adenylyl cyclase A, guanylyl cyclase, and phospholipase C by cAMP/CAR signaling in Dictyostelium absolutely requires G proteins, but, remarkably, using a series of Dictyostelium strains mutated for certain G protein gene(s), we and others have also described cAMP receptor signaling pathways (e.g. Ca+2 influx) that do not appear to require productive interaction with G proteins. Furthermore, cells that express only a single, temperature-sensitive G-beta subunit, continue to exhibit cAMP/CAR-regulated gene expression at a restrictive temperature. Since these transcriptional pathways are also GSK3-dependent, the results may suggest that CAR regulation of GSK3 does not require G proteins, and we speculate that Wnt regulation of GSK3 in other systems may also be G protein-independent. Mammalian GSK3 activity in vitro is acutely sensitive to tyrosine phosphorylation, but an in vivo regulatory pathway for this phosphorylation is not yet described. We have screened Dictyostelium cDNA expression libraries with antibodies to phosphotyrosine, and identified ZAK1, a kinase with a JAK-like organization. Similar to metazoan JAK tyrosine kinases that mediate cytokine response by activating STAT transcription factors, ZAK1 has two kinase homology domains. But ZAK1 is sufficiently diverged from JAK to suggest it is distinct. ZAK1 and JAKs have functional carboxyl-terminal tyrosine kinase domains, but while JAKs have inactive pseudokinase domains at their amino termini, the ZAK1 equivalent domain is a bona fide serine/threonine kinase. zak1-nulls have a global decrease in developmentally regulated phosphotyrosine, are morphologically deranged, and fail to complete terminal differentiation. zak1-nulls, like Dictyostelium gsk3-nulls, have a major defect in differentiating posterior, prespore cells. Consistent with this phenotype, zak1-nulls have reduced levels of GSK3 activity. Like CAR4, the RING/zipper adaptor protein rZIP inhibits prespore, but promotes prestalk differentiation. Using cell-specific markers and developmental chimaeras of wild-type and rZIP-nulls, we have shown that rZIP functions in cooperation with the cAMP receptors to control the graded accumulation of a secreted, diffusable factor that stimulates posterior (prespore) cell differentiation. Studies of cells that ectopically express the catalytic subunit or a dominant-negative regulatory subunit of the cAMP-dependent protein kinase (PKA) suggest a genetic interaction of rZIP with PKA that mediates an intracellular response to this gradient. We have also identified prespore and spore promoter segments that respond to this transcriptional activation gradient and localize prespore- and spore-specific gene expression. We have partially purified the secreted prespore differentiation factor(s) and continue to investigate the biochemical pathways that coordinate intracellular responses. We also seek to understand the mechanisms of 7-TM receptor signaling that act on far downstream targets and ultimately direct cell-specific gene expression. We have analyzed several developmentally regulated promoters and identified critical DNA elements and specific [e.g. GBF, CRTF] or global [e.g. CHD1] trans- acting factors. CAR1 gene expression is regulated by multiple modes of response to extracellular cAMP that are mediated by distinct promoters. At the early stages of Dictyostelium development, G protein-dependent signaling of CAR1 serves to autoactivate CAR1 gene expression. We have identified an essential element within the early CAR1 promoter, shown that it can impart cAMP-regulated expression to a heterologous, minimal promoter, and identified a nuclear DNA-binding protein that recognizes specifically this sequence. This putative cAMP receptor transcription factor (CRTF) was purified to homogeneity, and peptide sequences were determined and used for cDNA and gene isolation. CRTF is a novel, 879 amino acid, zinc finger- type protein with essential DNA-binding elements restricted to its carboxyl terminal 223 amino acids. CRTF is essential for induced CAR1 expression and normal development. The crtf-nulls are phenotypically similar to car1-nulls. While the early CAR1 promoter is only active during a very narrow developmental window, CRTF mRNA, protein and DNA-binding activity are detected constitutively, suggesting an acute, post-translational activation mechanism dependent upon a CAR1/G protein regulatory pathway.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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Sztalryd, Carole; Bell, Ming; Lu, Xinyue et al. (2006) Functional compensation for adipose differentiation-related protein (ADFP) by Tip47 in an ADFP null embryonic cell line. J Biol Chem 281:34341-8
Brzostowski, Joseph A; Kimmel, Alan R (2006) Nonadaptive regulation of ERK2 in Dictyostelium: implications for mechanisms of cAMP relay. Mol Biol Cell 17:4220-7
Xu, Guoheng; Sztalryd, Carole; Lu, Xinyue et al. (2005) Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway. J Biol Chem 280:42841-7
Londos, C; Sztalryd, C; Tansey, J T et al. (2005) Role of PAT proteins in lipid metabolism. Biochimie 87:45-9
Naude, Bronwen; Brzostowski, Joseph A; Kimmel, Alan R et al. (2005) Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT. J Biol Chem 280:25596-603
Khurana, Taruna; Brzostowski, Joseph A; Kimmel, Alan R (2005) A Rab21/LIM-only/CH-LIM complex regulates phagocytosis via both activating and inhibitory mechanisms. EMBO J 24:2254-64
Liu, Xunxian; Rubin, Jeffrey S; Kimmel, Alan R (2005) Rapid, Wnt-induced changes in GSK3beta associations that regulate beta-catenin stabilization are mediated by Galpha proteins. Curr Biol 15:1989-97
Kreppel, Lisa; Fey, Petra; Gaudet, Pascale et al. (2004) dictyBase: a new Dictyostelium discoideum genome database. Nucleic Acids Res 32:D332-3
Kimmel, Alan R; Parent, Carole A; Gough, Nancy R (2004) Teaching resources. Spatial and temporal dynamics of signaling components involved in the control of chemotaxis in Dictyostelium discoideum. Sci STKE 2004:tr3
Kimmel, Alan R; Firtel, Richard A (2004) Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response. Curr Opin Genet Dev 14:540-9

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